Ferlins are a novel class of membrane-bound proteins with six C2 domains. In other membrane associated proteins, C2 domains have been implicated in the regulation of membrane fusion by divalent cation and phospholipid binding, and through protein interactions. Mutations in the worm ferlin homolog, fer-1, produce defective membrane fusion. Mutations in dysferlin cause muscular dystrophy in humans. Dysferlin mutations produce an abnormal accumulation of subsarcolemmal vesicles in muscle. We identified myoferlin because of its high homology to dysferlin. We generated antibodies specific to dysferlin and myoferlin and found that each protein is associated with the plasma membrane. In prefusion myoblasts, myoferlin is highly expressed, while dysferlin is expressed in mature myotubes. We demonstrated that the C2A domain of dysferlin and myoferlin bound phosphotidylserine in a calcium-dependent manner. Furthermore, a mutation dysferlin that is associated with muscular dystrophy reduced calcium sensitive phospholipid binding. In muscle, membrane fusion is necessary for myoblast fusion to multinucleate myotubes. Membrane fusion is also required for membrane repair. We hypothesize that dysferlin and myoferlin regulate membrane fusion events in muscle. Moreover, we believe that defects in these ferlin proteins lead to abnormal membrane fusion. To elucidate the role of ferlin proteins in membrane fusion in muscle: Aim 1) We will test the calcium sensitive phospholipid binding properties of normal and mutant ferlin C2 domains. Aim 2) We will study the role of ferlin proteins in regeneration and repair in vivo using murine models of ferlin underexpression and ferlin overexpression. Aim 3) We will characterize a novel ferlin. Aim 4) We will search for ferlin- interacting proteins to determine additional proteins that regulate muscle membrane fusion. Significance: These experiments are designed to improve our understanding membrane fusion in muscle. Membrane fusion in muscle is relevant to diseases such as muscular dystrophy and also to the normal aging process of muscle. [unreadable] [unreadable]